The present invention relates generally to a method for expressing a gene in a microorganism, and more specifically, to a method for expression of a gene by transforming a host microorganism belonging to the genus Corynebacterium or Brevibacterium with a recombinant DNA constructed of a DNA fragment containing at least one gene to be expressed and a vector DNA in which at least one of the DNAs is foreign to the host microorganism. Heretofore, recombinant DNA technology has been established using primarily Escherichia coli as the host microorganism. So far the production of peptides such as somatostatin, insulin, human growth hormone, human interferon-.alpha. and human interferon-.beta. or vaccines such as foot-and-mouth disease vaccine has been reported, and Escherichia coli is considered to be adequate as a host microorganism for the expression of these highly physiologically-active peptides or vaccines. In order to achieve enhanced production, secretion out of the cells and glycosylation of a desired protein or to avoid contamination with intracellular toxins, host-vector systems in yeasts or Bacillus subtilis have also been developed.
For the production of physiologically active substances such as peptides, proteins and the like, the microorganisms mentioned above, for which recombinant DNA technology has already been established, may be satisfactorily employed. However, for the industrial mass production of substances such as amino acids, nucleic acids, vitamins, antibiotics and the like by recombinant DNA technology, those host microorganisms heretofor used are not applicable, and suitable technology for those microorganisms conventionally used for the production of the particular substance has to be developed.
Corynebacterium glutamicum was used first for the industrial production of amino acids. Subsequently, the industrial production of amino acids such as glutamic acid, lysine, alanine, histidine, tryptophan, tyrosine, phenylalanine, threonine, isoleucine, valine, leucine, glutamine, proline, arginine and the like has been developed using coryneform bacteria including those microorganisms classified in the genus Corynebacterium. As of the present, most amino acids are now commercially produced using microorganisms.
Therefore, application of recombinant DNA technology to these microorganisms is considered to be very important to improve the production of amino acids and the like.
Recombinant DNA technology generally consists of the following steps:
(1) Fragmentation of a DNA containing a desired gene with restriction endonucleases; PA1 (2) Linearization of a vector DNA with the same restriction endonuclease; PA1 (3) Construction of a recombinant DNA by mixing the DNA fragments with linearized vector plasmid mentioned above for annealing and ligating both DNAs with a DNA ligase; PA1 (4) Introduction of the recombinant DNA into a host microorganism (transformation); and PA1 (5) Selection and cloning of a recombinant containing the desired gene.
Successful construction of a recombinant strain is, of course, dependent upon the efficiency of each step. Therefore it is necessary to determine and improve the efficiency of each step to obtain transformants with a reasonable efficacy. Even if a desired gene is successfully introduced into a host, it is very difficult to express it because of various barriers to the expression of foreign genes. Kagaku to Seibutsu 18, 110-118 (1978).
Microorganisms belonging to the genus Corynebacterium or Brevibacterium have not yet been successfully used as a host to introduce desired genes or vectors foreign to the host and to express the desired genes. To develop recombinant DNA technology using host microorganisms belonging to the genus Corynebacterium or Brevibacterium, the construction of autonomously replicable vectors having selectable markers and suitable cloning sites for many genes is required as well as the establishment of efficient transformation systems. Moreover, a method for overcoming the barriers mentioned above is also required.
In furthermore of the foregoing, plasmid vectors autonomously replicable in microorganisms belonging to the genus Corynebacterium or Brevibacterium and having appropriate selectable markers and suitable cloning sites have been constructed by common inventors and highly efficient transformation systems have been developed. These are described in U.S. patent application Ser. No. 368,035 and U.S. Ser. No. 368,034, both filed Apr. 13, 1982 and Japanese Patent Application Nos. 58186/81, 58187/81 and 65777/81. It has now been found that when a DNA fragment containing a foreign gene involved in the biosynthesis of amino acids is inserted into such a plasmid vector by in vitro recombinant DNA technology (U.S. Pat. No. 4,237,224) and Corynebacterium glutamicum L-22 or its derivatives are transformed with the recombinant DNA by the transformation system mentioned above, it is possible to express the foreign gene in the host microorganism and increase the production of such useful substances such as amino acids and the like.